Nibbling within the nucleus: turnover of nuclear contents

As the site of gene expression and regulation, the nucleus is the control center of the cell. It might be thought that degradation of nuclear contents is strictly ‘off-limits,’ given the importance of the genetic information contained within the nucleus, but it has recently been reported that partial degradation of the nucleus may occur in yeast. Here we summarize the evidence for the degradation and quality control of proteins found with the nucleus and its compartments, and of nucleic acids that may occur under certain specific conditions. Only under certain special conditions such as differentiation of the lens are the entire nuclear contents degraded. Received 6 September 2006; received after revision 25 October 2006; accepted 13 December 2006     

The corticotropin-releasing factor (CRF) family of peptides as local modulators of adrenal function

Corticotropin-releasing factor (CRF), also termed corticotropin-releasing hormone (CRH) or corticoliberin, is the major regulator of the adaptive response to internal or external stresses. An essential component of the adaptation mechanism is the adrenal gland. CRF regulates adrenal function indirectly through the central nervous system (CNS) via the hypothalamic-pituitary-adrenal (HPA) axis and via the autonomic nervous system by way of locus coeruleus (LC) in the brain stem. Accumulating evidence suggests that CRF and its related peptides also affect the adrenals directly, i.e. not through the CNS but from within the adrenal gland where they form paracrine regulatory loops. Indeed, CRF and its related peptides, the urocortins (UCNs: UCN1, UCN2 and UCN3), their receptors CRF type 1 (CRF₁) and 2 (CRF₂) as well as the endogenous pseudo-receptor CRF-binding protein (CRF-BP) are all expressed in adrenal cortical, medullary chromaffin and resident immune cells. The intra-adrenal CRF-based regulatory system is complex and depends on the balance between the local concentration of CRF ligands and the availability of their receptors. Received 19 December 2006; received after revision 20 February 2007; accepted 26 March 2007     

Prion: the chameleon protein

From Creutzfeldt-Jakob disease (CJD) to variant CJD through Gerstmann-Str?ussler-Scheinker syndrome, kuru and fatal familial insomnia, the journey leading to current understanding of the basic aspects of human prion diseases has been full of unexpected, but often dramatic and always fascinating twists. Recent progress in modeling prion diseases and characterization of the various prion protein forms reveal that such a wide spectrum of the diseases is associated with the chameleon-like conformational features of prions.     

Pelizaeus-Merzbacher disease: Genetic and cellular pathogenesis

Pelizaeus-Merzbacher disease (PMD) and the allelic spastic paraplegia type 2 (SPG2) arise from mutations in the X-linked gene encoding myelin proteolipid protein (PLP). Analysis of mutations affecting PLP, the major protein in central nervous system myelin, has revealed previously unsuspected roles for myelinating glia in maintaining the integrity of the nervous system. The disease spectrum for PMD and SPG2 is extraordinarily broad and can be best understood by accounting not only for the wide range of mutations that can occur but also for the effects of PLP1 mutations on both cell autonomous and non-cell autonomous processes in myelinating cells. Appreciating the wide range of genetic and cellular effects of PLP1 mutations is important for patient and family counseling, understanding disease pathogenesis, and, ultimately, for developing future disease-specific therapies. Received 24 April 2006; received after revision 3 July 2006; accepted 9 October 2006     

Immunophilins: for the love of proteins

Immunophilins are chaperones that may also exhibit peptidylprolyl isomerase (PPIase) activity. This review summarizes our knowledge of the two largest families of immunophilins, namely cyclophilin and FK506-binding protein, and a novel chimeric dual-family immunophilin, named FK506- and cyclosporin-binding protein (FCBP). The larger members of each family are modular in nature, consisting of multiple PPIase and/or protein-protein interaction domains. Despite the apparent difference in their sequence and three-dimensional structure, the three families encode similar enzymatic and biological functions. Recent studies have revealed that many immunophilins possess a chaperone function independent of PPIase activity. Knockout animal studies have confirmed multiple essential roles of immunophilins in physiology and development. An immunophilin is indeed a natural ‘protein-philin’ (Greek ‘philin’ = friend) that interacts with proteins to guide their proper folding and assembly. Received: 7 May 2006; received after revision 3 July 2006; accepted 24 August 2006     

AFM观察小白鼠心肌和肝核DNA片段的基因移位

用原子力显微镜(简称AFM)直接观察、体外表达和体外转录等实验技术组合,观察到了心肌和肝的核DNA片段的基因;用磷酸缓冲液稀释,并用开关蛋白质等活性因子使其部分解离,促使核基因在核DNA片段内或核DNA片段间静态或动态移位,得到了对应核DNA片段中的相关基因,如LDH/DNA体外表达活性变化的LDH同功酶酶谱图。基因静态或动态移位均表达活性降低,且基因移位程度与其对应基因活性降低程度呈正相关性。展示了未来运用AFM和体外表达等实验技术组合研究核DNA片段的基因移位和对应基因突变机制的前景。     

小鼠脂多糖应答蛋白mlrpS的载体构建及融合蛋白表达

为构建小鼠mlrpS-cDNA基因原核、真核表达载体,大肠杆菌表达其融合蛋白。采用反转录-聚合酶链反应从经脂多糖刺激的鼠NIH3T3细胞cDNA中,扩增出编码mlrpS的cDNA。用限制性内切酶KpnⅠand XhoⅠ消化后,插入原核表达载体pTAT中,经酶切鉴定与测序证实后,转化大肠杆菌BL21（DE3）菌株。异丙基β—D硫代半乳糖苷（IPTG）诱导产生融合蛋白。经KpnⅠ和XhoⅠ酶切回收mlrpS-cDNA,插入pcDNA3．1载体中;pcDNA3．1-mlrpS再经KpnⅠ和ApaⅠ酶切后,插入到pEGFP—c1载体上,构建pEGFP-mlrpS融合的真核表达载体。构建mlrpS表达载体经测序证实,与GenBank登录的序列完全一致;双酶切鉴定证实,克隆基因正确插入载体pEGFP及pTAT;SDS—PAGE证实融合蛋白表达成功。说明：成功地构建了mlrpS原核、真核表达载体,成功正确表达了6His／mlrpS融合蛋白。     

Structure-function relationships of the polypyrimidine tract binding protein

The polypyrimidine tract binding protein (PTB) is a 58-kDa RNA binding protein involved in multiple aspects of mRNA metabolism including splicing regulation, polyadenylation, 3′end formation, internal ribosomal entry site-mediated translation, RNA localization and stability. PTB contains four RNA recognition motifs (RRMs) separated by three linkers. In this review we summarize structural information on PTB in solution that has been gathered during the past 7 years using NMR spectroscopy and small-angle X-ray scattering. The structures of all RRMs of PTB in their free state and in complex with short pyrimidine tracts, as well as a structural model of PTB RRM2 in complex with a peptide, revealed unusual structural features that provided new insights into the mechanisms of action of PTB in the different processes of RNA metabolism and in particular splicing regulation. Received 16 August 2007; received after revision 18 September 2007; accepted 2 October 2007     

Structural biology of the purine biosynthetic pathway

Purine biosynthesis requires ten enzymatic transformations to generate inosine monophosphate. PurF, PurD, PurL, PurM, PurC, and PurB are common to all pathways, while PurN or PurT, PurK/PurE-I or PurE-II, PurH or PurP, and PurJ or PurO catalyze the same steps in different organisms. X-ray crystal structures are available for all 15 purine biosynthetic enzymes, including 7 ATP-dependent enzymes, 2 amidotransferases and 2 tetrahydrofolate-dependent enzymes. Here we summarize the structures of the purine biosynthetic enzymes, discuss similarities and differences, and present arguments for pathway evolution. Four of the ATP-dependent enzymes belong to the ATP-grasp superfamily and 2 to the PurM superfamily. The amidotransferases are unrelated, with one utilizing an N-terminal nucleophileglutaminase and the other utilizing a triad glutaminase. Likewise the tetrahydrofolate-dependent enzymes are unrelated. Ancestral proteins may have included a broad specificity enzyme instead of PurD, PurT, PurK, PurC, and PurP, and a separate enzyme instead of PurM and PurL. Received 26 May 2008; received after revision 30 June 2008; accepted 9 July 2008     

甘蓝型油菜(Brassica napus)中一个未知功能蛋白基因的cDNA克隆

随着现代分子生物学的发展,许多物种,特别是拟南芥等模式生物的整个基因组序列以及其中一部分基因的功能已经比较清楚,但仍然存在大量功能未知的基因有待进一步研究,从已知片段出发,利用cDNA末端快速扩增（rapid amplification of cDNA ends,RACE）技术钓取基因全长,并结合生物信息学技术预测蛋白质结构,是研究未知基因功能的一种重要手段．